Separation and purification of bovine serum albumin binders from Fructus polygoni orientalis using off-line two-dimensional complexation high-speed counter-current chromatography target-guided by ligand fishing

• (BSA-Fe3O4MNPs)-HPLC–MS/MS was developed to screen bioactive compounds from Fructus polygoni orientalis.
• Off-line 2D HSCCC with several operation modes including Recycling HSCCC, Normal-Phase HSCCC and Reverse-Phase HSCCC was used to isolate target compounds.
• Copper ion as a complexation agent was introduced into HSCCC two-phase solvent systems.
• Complexation HSCCC behavior of seven compounds was explained by investigating their chelating power with copper ion.

In this study, off-line two-dimensional (2D) complexation high speed counter-current chromatography (HSCCC) was developed for the separation of bovine serum albumin (BSA) binders from the ethyl acetate extract of Fructus polygoni orientalis. Target-guided strategy of BSA functionalized iron oxide magnetic nanoparticles coupled with high performance liquid chromatography–tandem mass spectrometry ((BSA-Fe3O4 MNPs)-HPLC–MS/MS) experiment was proposed. In the orthogonal separation system, a Normal-Phase HSCCC with 0.01 mol/L copper ion as complexation agent in the aqueous phase was employed for the first dimension and Recycling HSCCC, Reverse-Phase HSCCC with 0.1 mol/L copper ion were used for the second dimension in parallel. Including two pairs of cis-trans isomers, seven BSA binders including 3,5,7-Trihydroxychromone (1), taxifolin (2), N-cis-paprazine (3), N-cis-feruloyltyramine (4), N-trans-paprazine (5), N-trans-feruloyltyramine (6) and an unidentified compound (7) were obtained. The purities of these seven compounds were all over 95.0% as determined by HPLC. The complexation HSCCC behaviors of seven compounds were also investigated by studying their relationship with copper ion. Results showed that the combinative method using (BSA-Fe3O4 MNPs)-HPLC and HSCCC is a quick, efficient, and reproductive technique to isolate potentially bioactive compounds from the complex mixture system of natural products. And the usage of off-line 2D-HSCCC and introduction of chelating metal ion into solvent system are effective ways to implement HSCCC separations in complex samples.